Biosensors for detecting macromolecular biomolecules are increasingly gaining in importance. [1], [2] and [5] to [12] describe DNA sensors known from the prior art.
An important sensor type, particularly in the case of fully electronic DNA sensor chips, is so called redox cycling. The basics of redox cycling are described in [3], [4]. In redox cycling, macromolecular biopolymers are identified electronically on surfaces by detecting electric currents caused by means of redoxactive markings.
FIG. 1A, FIG. 1B show a redox cycling sensor arrangement 100 in accordance with the prior art.
The redox cycling sensor arrangement 100 has two gold working electrodes 101, 102 that are formed on a substrate 103. DNA capture molecules 104 having a prescribed sequence are immobilized on each working electrode 101, 102. The immobilization is performed, for example, in accordance with the so called gold-sulfur coupling. Furthermore, an analyte 105 to be examined is introduced into the redox cycling sensor arrangement 100. The analyte 105 can be, for example, an electrolytic solution with different DNA molecules.
If the analyte 105 contains first DNA half strands 106 with a sequence that is not complementary to the sequence of the DNA capture molecules 104, these first DNA half strands 106 do not hybridize with the DNA capture molecules 104 (see FIG. 1A). It is said there is a “mismatch” in this case.
If, by contrast, the analyte 105 contains second DNA half strands 107 with a sequence that is complementary to the sequence of the DNA capture molecules 104, these second DNA half strands 107 hybridize with the DNA capture molecules 104. It is said there is a “match” in this case. Otherwise expressed, a DNA half strand 104 of a prescribed sequence is respectively only capable of hybridizing selectively with a very specific DNA half strand, specifically with the DNA half strand with the sequence that is complementary to the respective capture molecule.
As FIG. 1B shows, the second DNA half strands 107 to be detected include a redoxactive marking 108. After the hybridization of the second DNA half strands 107 to be detected and having the DNA capture molecules 104, in the presence of suitable additional molecules 109 (for example para-aminophenylphosphate, p-APP) the redoxactive marking 108 (for example an enzyme label such as an alkaline phosphatase, for example) is used to initiate a cycle of oxidations and reductions of constituents of the additional molecules 109 that leads, to the accompaniment of interaction with the gold electrodes 101, 102, to the formation of reduced molecules 110 (for example para-aminophenol) or oxidized molecules 111 (for example quinoneimine). The cycle of oxidations and reductions leads to an electric ring current that enables the second DNA half strands 107 to be identified.
Consequently, in the case of a binding event between a particle to be detected and a capture molecule, a redoxactive species is generated in the redox cycling method by means of an enzyme label (for example an alkaline phosphatase), for example by converting para-aminophenylphosphate (p-APP) contained in an electrolyte into para-aminophenol. Since new redoxactive species are continuously generated, this leads to a rise in the electric current between the two electrodes.
An oxidizing electrical potential is required at the first working electrode 101, which is also denoted as generator electrode. A reducing electrical potential is required at the second working electrode 102, which is also denoted as collector electrode.
FIG. 2 shows an interdigital electrode arrangement 200 that is known from the prior art and has two working electrodes interlocking in the form of fingers, specifically a generator electrode 201 and a collector electrode 202. A reference electrode 203 and a counter electrode 204 are also shown. The electrodes 201 to 204 are formed on a substrate 205. An electrolytic analyte (not shown) that is coupled to the electrodes 201 to 204 can be applied to the interdigital electrode arrangement 200. The reference electrode 203 provides the electrical potential of the electrolytic analyte to an inverting input of a comparator 206 that compares it with the desired electrical potential at the noninverting input of the comparator 206.
In the event of a deviation of the electrical potential of the reference electrode 203 from the desired potential, the counter electrode 204 is driven via an output of the comparator 206 so that, if needed, it resupplies electric charge carriers in order to maintain the desired electrical potential of the electrolyte. Evidently, the reference electrode 203 forms a potentiostat device together with the comparator 206. The electrical potentials at the working electrodes 201, 202 are set relative to the reference voltage. Electric sensor currents of the generator electrode 201 or the collector electrode 202 are detected by means of first and second ammeters 207, 208 which contain information relating to a sensor event that may have occurred.
Also known from the prior art is a sensor array in the case of which a plurality of interdigital electrode arrangements 200 are interconnected, for example in the form of a matrix. Components 203, 204, 207, 208 for a number of sensor fields can be jointly provided therein.
If a sensor event occurs at a sensor field of such a sensor array, reduced molecules 110 or oxidized molecules 111 are formed. It is desired for these charged particles to be electrically detected at the working electrodes 201 and 202. However, these charged particles are frequently exposed to diffusion in an analyte and can undesirably diffuse to an adjacent sensor field (or an adjacent pixel) where they generate an undesired electric interference signal that falsifies the measurement event or generates a sensor signal at false sensor electrodes without a sensor event having taken place at these sensor electrodes.
An attempt is made in accordance with the prior art to meet this problem by selecting the measuring time to be so short that no undesired diffusion comes to bear, or remains negligibly small. This procedure is, however, disadvantageous, since it is then impossible to utilize the entire dynamics of the redox process for measuring. Otherwise expressed, given a measuring time selected to be too short, many electrically charged particles that are a consequence of a sensor event are lost in the measurement. Consequently, the sensitivity of identification is reduced or the signal-to-noise ratio is worsened.
In accordance with the prior art, [5] describes a method for detecting molecules or molecule complexes with the aid of an arrangement that has ultramicroelectrode arrays whose electrode structures are arranged so closely next to one another that the distances between the various structures lie in the ultramicro range. Use is made, in particular, of the effect that adjusting electric fields can be generated between closely adjacent electrodes, and the resulting current is influenced chiefly by the detected molecules and molecule complexes in the space near the electrodes. This influencing effect can be formed by diffusion, by accumulation or binding of the species to be measured.
[6] describes an electrical sensor array that has a number of sensor positions that respectively include at least two microelectrodes. This array can be used for simultaneously detecting various molecular substances from mixtures of substances in an electrochemical fashion. In particular, it is possible to address individual sensor positions individually.
[7] shows interdigital electrode arrangements on flexible substrates for the purpose of measuring the electrical behavior of substances. The arrangements contain electrode structures with a working electrode and a counter electrode.
[8] describes a biosensor array and a method for operating a biosensor array. The biosensor array has at least one first and one second signal line that are coupled to at least two of the biosensor fields. This provides a plurality of biosensor fields with joint signal lines for driving and detecting.
[9] describes a sensor for the qualitative and quantitative determination of (bio)organic oligomers and polymers. It is provided here at least one detection electrode on which capture molecules are immobilized for hybridization with organic oligomers and polymers that are to be determined, as well as at least two attraction electrodes on which no capture molecules are located. The detection electrode is arranged between the attraction electrodes in such a way that an analyte which possibly contains the chemical compounds to be detected and is applied to the sensor arrangement is moved away over the detection electrode by changing electric fields at the attraction electrodes, as a function of the type and size of the electric fields.
[10] shows a biosensor chip that has a first and a second electrode. The first electrode has a holding region for holding probe molecules that can bind macromolecular biopolymers. Also provided is an integrated electric differentiator circuit with which an electric current generated during a reduction/oxidation recycling operation is detected and can be differentiated with respect to time.
[11] describes an arrangement for an electrochemical analysis method and use thereof. This arrangement has an electrode system composed of at least three electrodes, at least one working electrode, one counter electrode and one reference electrode being present. The reference electrode is arranged in such a way that it is adjacent to at least subregions of the two further electrodes. It is preferably spaced apart equally from these subregions.
[12] describes a micro-multielectrode arrangement for the electrochemical measurement and generation of electroactive species, the electrodes being arranged on a carrier, in this case. One inner electrode and at least two further electrodes are provided, the inner electrode being connected as a reference electrode, and the further electrodes at least partially surrounding the inner electrode in the projection onto the carrier. The at least two further electrodes are a measuring electrode and a counter electrode, the counter electrode being arranged at a greater central distance from the reference electrode than the measuring electrode.